Application of paired end short read library to identify promoter region

[Moondust]

Hi all,

I just got to know about the paired-end short reads sequencing. Can someone help me clarify if i have understood it correctly?

From what i understand, in one sheared genomic fragment, (eg 5'AdaptorTTTTTTT..........CCCCCCC3' and 5' adaptor GGGGGG.........AAAAAAA), only 35bp can be read from both ends.

Does that includes the adaptor sequence? i.e (5'Adaptor sequence +TTTT...til it makes up to 35bp) and 5'AdaptorGGGGG.. for complementary sequence read.

Thus based on these short fragments they are aligned and there may be unknown sequences between the two adaptors.

Suppose if i want find a promoter region of a GOI, what I can do is to BLAST my GOI and find an adaptor sequence that is in my GOI region and another outside of the gene in the 5'UTR?

From there I design primers to amplify the region I want (eg 5'TTTTTT and 5'GGGGGG if I take the above mentioned gene sequence as an example?)

Please correct me if i have misuderstood the technology. Thanks heaps.

[elaney_k]

Hi Moondust,

The adapters are not sequenced, only the DNA between them is. If you are only interested in a particular genomic region then yes you can design primers and amplify by PCR, this PCR product can then be used as input for library generation and sequencing instead of DNA however you would need to be targetting a large region or many regions or indexing your samples to make it economical to use the next-gen technologies.

Elaine